Throughout this application, various publications are referenced by Arabic numerals. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
The life cycle of animal viruses is characterized by a series of events that are required for the productive infection of the host cell. The initial step in the replicative cycle is the attachment of the virus to the cell surface, which attachment is mediated by the specific interaction of the viral attachment protein (VAP) to receptors on the surface of the target cell. The differential pattern of expression of these receptors is largely responsible for the host range and tropic properties of viruses. In addition, an effective immune response against many viruses is mediated through neutralizing antibodies directed against the VAP. The interaction of the VAP with cellular receptors and the immune system therefore plays a critical role in infection and pathogenesis of viral disease.
The human immunodeficiency virus type 1 (HIV-1) infects primarily helper T lymphocytes, dendritic cells, and monocytes/macrophages--cells that express surface CD4--leading to a gradual loss of immune function. This loss of function results in the development of the human acquired immunodeficiency syndrome (AIDS) (1). The initial phase of the HIV-1 replicative cycle involves the high-affinity interaction between the HIV-1 exterior envelope glycoprotein gp120 and cell surface CD4 (K.sub.d approximately 4.times.10.sup.-9 M) (2). Several lines of evidence demonstrate the requirement of this interaction for viral infectivity. The introduction into CD4.sup.- human cells of cDNA encoding CD4 is sufficient to render otherwise resistant cells susceptible to HIV-1 infection (3). In vivo, viral infection appears to be restricted to cells expressing CD4, indicating that the cellular tropism of HIV-1 is largely determined by the pattern of cellular expression of CD4. Following the binding of HIV-1 gp120 to cell surface CD4, viral and target cell membranes fuse by a mechanism that is poorly understood, resulting in the introduction of the viral capsid into the target cell cytoplasm (4).
Mature CD4 has a relative molecular mass (Mr) of 55 kDa and consists of an N-terminal 372-amino acid extracellular domain containing four tandem immunoglobulin-like regions (V1-4), followed by a 23-amino acid transmembrane domain and a 38-amino acid cytoplasmic segment (5, 6). In experiments using truncated sCD4 proteins, it has been shown that the determinants for high-affinity binding to HIV-1 gp120 lie solely within the N-terminal immunoglobulin-like domain (V1) (7-9). Mutational analysis of V1 has defined a discrete binding site (residues 38-52) that comprises a region structurally homologous to the second complementarity-determining region (CDR2) of immunoglobulin genes (9).
The production of large quantities of sCD4 has permitted a structural analysis of the two N-terminal immunoglobulin-like domains (V1V2). The structure determined at 2.3 angstrom resolution reveals that the molecule has two tightly-associated domains, each of which contains the immunoglobulin-fold connected by a continuous beta strand. The putative binding sites for monoclonal antibodies, class II major histocompatibility complex (MHC) molecules, and HIV-1 gp120, as determined by mutational analyses, map on the molecular surface (10, 11).
The HIV-1 envelope gene env encodes an envelope glycoprotein precursor, gp160, which is cleaved by cellular proteases before transport to the plasma membrane to yield gp120 and gp41. The membrane-spanning glycoprotein, gp41, is non-covalently associated with gp120, a purely extracellular glycoprotein. The mature gp120 molecule is heavily glycosylated (approximately 24 N-linked oligosaccharides), contains approximately 480 amino acid residues with 9 intra-chain disulfide bonds (12), and projects from the viral membrane as a dimeric or multimeric molecule (13).
Mutational studies of HIV-1 gp120 have delineated important functional regions of the molecule. The regions of gp120 that interact with gp41 map primarily to the N- and C-termini (14). The predominant strain-specific neutralizing epitope on gp120 is located in the 32-34 amino acid residue third variable loop, herein referred to as the V3 loop, which resides near the center of the gp120 sequence (15). The CD4 binding site maps to discontinuous regions of gp120 that include highly conserved or invariant amino acid residues in the second, third, and fourth conserved domains (the C2, C3, and C4 domains) of gp120 (16). It has been postulated that a small pocket formed by these conserved residues within gp120 could accommodate the CDR2 loop of CD4, a region defined by mutational analyses as important in interacting with gp120 (17).
HIV-1 gp120 not only mediates viral attachment to surface CD4 molecules, but also serves as the major target of antibodies which neutralize non-cell-associated virus and inhibit cell to cell viral transmission.
There are two major classifications of HIV-1-neutralizing antibodies: type-specific and group-common (15). Type-specific neutralizing antibodies primarily recognize linear determinants in the highly variable V3 loop of gp120. These antibodies act by inhibiting fusion between HIV-1 and the target cell membrane, and generally neutralize only a particular isolate of, or closely related strains of, HIV-1. Sequence variation within the V3 loop, as well as outside of this region, permits viruses to escape neutralization by anti-V3 loop antibodies. In contrast, group-common neutralizing antibodies primarily recognize discontinuous or conformational epitopes in gp120, and possess the ability to neutralize a diverse range of HIV-1 isolates. These broadly neutralizing antibodies often recognize a site on gp120 which overlaps the highly conserved CD4-binding site, and thus inhibits gp120-CD4 binding.
A structural relationship has been demonstrated between the V3 loop and the C4 region of gp120 which region constitutes both part of the CD4 binding site and part of the conserved neutralization epitopes. It was observed that deleting the V3 loop resulted in significantly increased binding of a panel of broadly neutralizing hMoAbs (neutralizing human monoclonal antibodies) to the CD4 binding site (18).
A major goal in AIDS vaccine development is to develop a vaccine able to protect a subject against the numerous genetic variants of HIV-1 that infect humans. Although cell-mediated immune responses might serve to control infection in HIV-1-infected individuals, several lines of evidence demonstrate that protection against infection is mainly mediated by neutralizing antibodies directed against gp120. Early experiments showed that immunization of chimpanzees with recombinant gp120 induced a protective immune response against challenge with the homologous HIV-1 strain (17). This protection correlated with the presence of high-titer neutralizing antibodies against the V3 loop of gp120. In addition, passive immunization of chimpanzees with a V3-loop neutralizing monoclonal antibody resulted in protection against challenge with the homologous HIV-1 strain (19). Although protection against challenge was demonstrated in these two experiments, recent studies have questioned the clinical relevance of these findings. For example, these neutralizing antibodies recognize the V3 loop determinants of a single strain, and not conserved or discontinuous epitopes. Thus, these antibodies lack the ability to neutralize the broad spectrum of HIV-1 strains present in an HIV-1 population. Furthermore, the challenge virus was the homologous HIV-1 laboratory adapted LAI (HTLV-IIIB) strain and not one of the primary isolates that contain considerable gp120 sequence heterogeneity. Since these experiments showed that gp120 subunit vaccination induces an immune response effective against only the homogeneous HIV-1 strain used as an antigen, it is unlikely that the vaccination regimens used in these studies would be useful in humans.
Individuals infected by HIV-1 typically develop antibodies that neutralize the virus in vitro, and neutralization titers decrease with disease progression (19). Analysis of sera from HIV-1-infected humans indicates that type-specific neutralizing antibodies appear early in infection. Later in the course of infection, a more broadly neutralizing antibody response develops. However this antibody response is of significantly lower titer and/or affinity. Fractionation studies of HIV-1 antibody-positive human sera reveal that the type-specific neutralizing activity is primarily directed against linear determinants in the V3 loop of gp120 (20). There was no correlation found among antibodies between the ability to neutralize divergent HIV-1 isolates and reactivity to the V3 loop of these isolates. In contrast, the broadly neutralizing antibodies present in HIV-1 antibody-positive human sera primarly recognize discontinuous epitopes in gp120 which overlap the CD4-binding site and block gp120-CD4 binding. In other words, the broadly neutralizing activity of neutralizing antibodies is not merely the result of additive anti-V3 loop reactivities against diverse HIV-1 isolates which appear during infection.
Recently, several groups have generated human monoclonal antibodies (hMoAbs) derived from HIV-1 infected individuals which possess type-specific or group-common neutralizing activities (17). The type-specific neutralizing hMoAbs were found to recognize linear determinants in the V3 loop of gp120. In contrast, the group-common neutralizing hMoAbs generally recognize discontinuous epitopes which overlap the CD4-binding site and block gp120-CD4 binding.
The V3 loop is a highly immunodominant region of gp120 which partially interacts with the CD4-binding region. The presence of the V3 loop region on gp120 may skew the humoral immune response away from producing antibodies which specifically bind to the CD4-binding domain of gp120. Furthermore, the advantages of removing the V3 loop to expose the CD4-binding domain of gp120 to the immune system would be countered by the fact that the exposed CD4-binding site would still have a high affinity for cell surface CD4. In other words, a mutant gp120 protein missing only the V3 loop would quickly bind to CD4+ cells and would thus be hampered in generating an immune response against the exposed CD4-binding site.
The subject invention provides a mutant HIV-1 gp120 envelope glycoprotein which overcomes both the problems of V3 loop immunodominance and of the high affinity to CD4. The subject invention further provides vaccines comprising the mutant HIV-1 gp120 envelope glycoprotein, antibodies which specifically bind to the CD4-binding site of HIV-1 gp120 envelope glycoprotein, pharmaceutical compositions comprising these antibodies, and methods of using these vaccines and compositions to treat or prevent HIV-1 infection.